The present invention relates to fluorescence detection method, detection apparatus and fluorescence detection program and more particularly, to a technology for automating fluorescence photometric method and fluorescence photometric apparatus.
In a conventional fluorescence detection apparatus, a sample labeled with a fluorescent material is irradiated with an exciting light ray of an excitation wavelength optimized for the labeled fluorescent material and a fluorescent luminescence ray associated with a wavelength, at which the highest fluorescence intensity is exhibited in a wavelength band of a luminescence area characteristic of the labeled fluorescent material, is detected to quantitatively determine the sample. When detecting an unknown fluorescent material within a detection range of fluorescent analyzer, a method as disclosed in JP-A-58-21143 (Patent Document) is available according to which an analysis is made within a detectable range by changing the sensitivity of an amplifier.
In carrying out fluorescence detection in respect of a fluorescent material having an unknown concentration or a sample labeled with the fluorescent material having an unknown concentration, the wavelength band of exciting light wavelengths or the sensitivity of an amplifier is changed as indicated in the Patent Document, or alternatively the concentration of the sample is measured and the sample is diluted or enriched in accordance with a measured concentration, thereby assuring detection within the detection range. With the sensitivity of the amplifier changed for the sake of confining detection intensities in the detection range, there arises a problem that the detection waveform is distorted as shown in FIG. 11 or the difference in detection sensitivity occurs between a standard sample and the sample to be inspected (inspection sample). For example, in determining a concentration of the inspection sample in terms of a relative value to that of the standard sample from an area of a detected waveform, difficulties are encountered in accurately settling the detected waveform area. Further, in setting up a base line (background) during determination of a concentration of the inspection sample and a relative value thereof to that of the standard sample, the positioning of a base line by drawing the line from start point to end point for an detected waveform is difficult due to the method that as the amplification sensitivity is switched from one level to another, the start point or end point of detection value changes before and after its switch.
Further, for adjustment of the concentration of a sample, processing procedures are needed including the steps of detecting a concentration of the sample and adjusting the concentration of the sample as well as adjusting again the concentration of a sample which is determined, after detection, as deviating from the detection range, thus being considered to be problematic from the standpoints of the complexity of operation and the throughput. In addition, in the case of an apparatus for detecting many samples of different concentrations, different concentration adjustment operations are necessary for a host of samples and the operations can be automated only at the cost of the provision of a mechanism having a system for detecting concentrations, a pipetting device capable of adjusting individual samples separately and a space made room for dilution and enrichment, causing many problems leading to high costs, degraded throughput and augmented scale for the automated pipetting device.